Construction and evaluation of DNA vaccine encoding Crimean Congo hemorrhagic fever virus nucleocapsid protein, glycoprotein N-terminal and C-terminal fused with LAMP1.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hemorrhagic fever in humans and is mainly transmitted by ticks. There is no effective vaccine for Crimean-Congo hemorrhagic fever (CCHF) at present. We developed three DNA vaccines encoding CCHFV nucleocapsid protein (NP), glycoprotein N-terminal (Gn) and C-terminal (Gc) fused with lysosome-associated membrane protein 1 (LAMP1) and assessed their immunogenicity and protective efficacy in a human MHC (HLA-A11/DR1) transgenic mouse model. The mice that were vaccinated three times with pVAX-LAMP1-CCHFV-NP induced balanced Th1 and Th2 responses and could most effectively protect mice from CCHFV transcription and entry-competent virus-like particles (tecVLPs) infection. The mice vaccinated with pVAX-LAMP1-CCHFV-Gc mainly elicited specific anti-Gc and neutralizing antibodies and provided a certain protection from CCHFV tecVLPs infection, but the protective efficacy was less than that of pVAX-LAMP1-CCHFV-NP. The mice vaccinated with pVAX-LAMP1-CCHFV-Gn only elicited specific anti-Gn antibodies and could not provide sufficient protection from CCHFV tecVLPs infection. These results suggest that pVAX-LAMP1-CCHFV-NP would be a potential and powerful candidate vaccine for CCHFV.

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers.

Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library.

Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.

Cellular immunogenicity of a multi-epitope peptide vaccine candidate based on hepatitis C virus NS5A, NS4B and core proteins in HHD-2 mice.

To develop a vaccine against hepatitis C virus (HCV), a multi-epitope peptide was synthesized from nonstructural proteins containing HLA-A2 epitopes inducing mainly responses in natural infection. The engineered vaccine candidate, VAL-44, consists of multiple epitopes from the HCV NS5A, NS4B and core proteins. Immunization with the VAL-44 peptide induced higher CTL responses than those by the smaller VL-20 peptide. VAL-44 induced antigen-specific IFN-γ-producing CD4+ T cells and CD8+ T cells. VAL-44 elicited a Th1-biased immune response with secretion of high amounts of IFN-γ and IL-2, compared with VL-20. These results suggest that VAL-44 can elicit strong cellular immune responses. The VAL-44 peptide stimulated IFN-γ production from viral-specific peripheral blood mononuclear cells (PBMCs) of patients infected with HCV. These results suggest that VAL-44 could be developed as a potential HCV multi-epitope peptide vaccine.

The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs.

BACKGROUND: Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71. METHODS: Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2'-modified (2'-O-methylation or 2'-fluoro modification) siRNAs were designed to target highly conserved 5' untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs. RESULTS: Transfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5' UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2'-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts. CONCLUSION: Sequences were identified within the highly conserved 5' UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2'-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.

[Cloning of the variable region genes from hybridoma against HAAH and then construction and expression of anti-HAAH scFv].

AIM: Construction and expression of anti-HAAH single chain variable fragment (scFv) by cloning of the variable region genes from anti-HAAH hybridoma cells G3/F11. METHODS: Total RNA was extracted from hybridoma cells G3/F11. By RT-PCR, murine V(H) and V(L) genes of mAb were amplified respectively. Then, They were assembled into V(H)-linker-V(L) scFv template by SOE-PCR and anti-HAAH scFv was express in E.coli by constructed pHEN 1-anti-HAAH vector. The expression of anti-HAAH scFv were detected by SDS-PAGE and Western blotting and the binding activity were demonstrated by ELISA. RESULTS: The analysis of DNA sequencing shown that the full-length of constructed scFv gene was 744 bp, encoding 248 amino acids. Moreover, the V(H) and V(L) genes were functional antibody variable region genes, as there were four FRs and three CDRs in both of them. By SDS-PAGE and Western blotting, the expression level of anti-HAAH scFv were detected. The expression level of pHEN 1-anti-HAAH scFv, which was expressed in E.coli HB2151, was 7.8% in total E.coli protein and were existed in soluble protein mainly. By indirect ELISA detection with HAAH protein, the binding activity of soluble anti-HAAH scFv was very well. CONCLUSION: The murine V(H) and V(L) genes of mAb against HAAH have been cloned successfully and anti-HAAH scFv have been constructed and expressed. Besides, the scFv could be further studied about their biological activity and application, due to their high affinity shown in preliminary detection.

[Immune response in BALB/c mice immunized with BCG expressing HBV truncated C gene and preS1 gene].

AIM: To study expressing of HBV truncated core and preS1 protein in BCG and to explore the effect of humoral and cellular immune response stimulated by the recombinant BCG. METHODS: A shuttle vector was constructed and was transformed into BCG which including truncated Core gene and preS1 gene of HBV. The recombinant BCGs were analyzed with SDS-PAGE and Western blot. The three groups of BALB/c mice were immunized with saline, BCG, BCG-pDE22 and BCG-pDE22-CS1 respectively. Then the antibody titer and CTL effects were evaluated. RESULTS: Compared with the control group, the recombinant BCG could express a new protein band of 24 kD which was consistent with the size of CS1 fused protein which displayed a good antigen-binding property by Western blot analysis. The antibody titer significantly increased and CTL effect apparently enhanced in the recombinant BCG immunized group. CONCLUSION: BCG could be as live vector which carrying HBV related genes, which developing a novel vaccine against HBV.

[A comparative study on the properties of trichosanthin before and after site-directed PEGylation].

OBJECTIVE: To construct PEGylated trichosanthin (TCS) mutein and analyze its bioactivities, immunogenicity, acute toxicity, and pharmacokinetics. METHODS: The potential antigenic determinant site YFF81-83 in the molecule of TCS was selected to undergo site-directed mutagenesis. Thus, a TCS mutein named TCS(YFF81-83ACS) was constructed and expressed in Escherichia coli of the line BL21 (DE3). Wild TCS (wTCS), TCSY(FF81-83ACS), and PEGylated TCS(YFF81-83ACS) (PEG- TCS(YFF81-83ACS)) of different concentrations were incubated with the supercoiled plasmid pUC19 to detect the DNAse activity, mixed with rabbit reticulocyte lysate to detect the ribosome inactivation activity, subcutaneously injected into 6 mice respectively to measure the serum IgG and IgE levels, intravenously injected into mice to observe the toxicity, and intravenously injected into SD rats to observe its -plasma half-life. RESULTS: The DNAse activity of the PEG-TCS(YFF81-83ACS) was similar to that of the wTCS. The ribosome inactivation activity of the PEG-TCS(YFF81-83ACS) was 1/9-1/8 of that of the wTCS (P < 0.05). The serum IgE and IgG levels of the PEG-TCS(YFF81-83ACS) were both significantly lower than those of the wTCS (both P < 0.05). The LD50 of the PEG-TCS(YFF81-83ACS) was 1.8 times that of the wTCS (P < 0.05). The mean residence time and plasma half-life of the PEG-TCS(YFF81-83ACS) were significantly increased and its plasma clearance was significantly decreased (all P < 0.05). CONCLUSION: Site-directed mutagenesis and PEGylation of TCS provide a new approach for reconstructing TCS.

Production and characterization of monoclonal antibody specific for NS3 helicase of hepatitis C virus.

Hepatitis C virus (HCV) infection is the major etiological agent of chronic hepatitis, which leads to liver cirrhosis and hepatocellular carcinomas. HCV NS3 helicase is a promising target of anti-virus therapy. In this report, we discuss a strategy to generate monoclonal antibodies (MAbs) of the HCV NS3 helicase, and investigate its potential characteristic. Our results showed the production of MAbs against NS3 helicase, which could specifically recognize the native NS3 helicase in transiently transfected cells in the immunofluorescence experiment. The resultant MAbs were used as the first antibody in Western blot analyses, and observed the specific band that defines the NS3 helicase. Likewise, one MAb could inhibit the NS3 helicase enzymatic activity distinctly in the NS3 helicase-mediated DNA-unwinding assay. To conclude, these antibodies may be useful to generate specific diagnostic tools for HCV infection and may also be developed for potential therapeutics.

The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.

[Construction of eukaryotic expression plasmid expressing siRNA targeting HLA-G gene and detection of its specific downregulation effect].

AIM: To investigate the effect of HLA-G siRNA on the protection of trophoblast cells from NK cell lysis, an HLA-G-targeting siRNA (small interfering)-expressing plasmid was constructed. METHODS: HLA-G siRNA plasmid, pSuppressor-U6-neo-HLA-G, was constructed and transfected to JEG-3 cells. The level of messenger RNA and protein of HLA-G in the transfected cells was detected by RT-PCR and Western blot. NK-92MI cells were co-cultured with pSuppressor-U6-neo-HLA-G transfected JEG-3 cells. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay. RESULTS: Downregulation of HLA-G in HLA-G siRNA plasmid transfected JEG-3 cells was confirmed by RT-PCR and Western blot. Compared with JEG-3 cells without HLA-G without HLA-G siRNA plasmid transfection, the transfected JEG-3 cells showed higher lytic activity. CONCLUSION: HLA-G siRNA can increasing NK cytolysis against JEG-3 cells. HLA-G plays a role in preventing of trophoblast cells from NK cytotoxicity.

[Construction of eukaryotic expression vector expressing hepatitis C virus NS5B and EGFP fusion protein and establishment of stable transfected HepG2 cell line].

OBJECTIVE: To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line. METHODS: The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained. RESULTS: The eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained. CONCLUSION: The stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.

[Identification of oligopeptides mimicking the virus attachment protein of hantaan virus].

OBJECTIVE: To identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus. METHODS: The monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM). RESULTS: The conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105. CONCLUSION: G2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.

[Preparation of specific monoclonal antibody against nucleocapsid protein of SARS coronavirus].

AIM: To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein. METHODS: N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb. RESULTS: DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the M(r) of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as primary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein. CONCLUSION: The prepared recombinant N protein and mAbs against N protein lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.